Rapid real time PCR to distinguish between high risk human papillomavirus types 16 and 18

Aims-To assess the validity and practicality of real time polymerase chain reaction (PCR) for human papillomavirus (HPV) testing in combination with l iquid based cytology samples for cervical screening. Methods-Real time PCR using consensus (GP5+/6+) and type specific primers w as developed to detect genital HPV types. This provides rapid, efficient am plification followed by denaturation of the product and computer analysis o f the kinetics data that are generated. Liquid based cytology samples were obtained from attending routine cervical screening clinics. DNA was extract ed from the residual cellular suspension after cytology using spin columns. Results-Real time PCR successfully distinguished between HPV-16 and HPV-18 on the basis of amplification with consensus primers followed by DNA meltin g temperature (T-m) analysis. Sensitivities of one to 10 copies of HPV-16 ( mean T-m = 79.4 degreesC; 2 SD, 0.8) and four to 40 copies of HPV-18 (mean T-m = 80.4 degreesC; 2 SD, 0.4) were obtained. In a mixed population of SiH a and HeLa cells containing known copy numbers of HPV-16 and HPV-18 genomes , HPV-16 and HPV-18 products were clearly separated by T-m, analysis in mix tures varying from equivalence to 1/1000. Together with detailed melt analy sis, type specific primers from the same region of the L1 gene confirmed th e differential ability of this system. The method was applied to 100 liquid based cytology samples where HPV status using conventional GP5+/6+ PCR was already known. There was 95% agreement between the methods, with 55 positi ves detected by conventional PCR and 59 with real time FCR. The method was then tested on 200 routine liquid based cytology samples. Approximately 10% were positive by real time PCR, most of which were classified as HPV-16 by detailed melt analysis. Thirteen (6.8%) HPV positives were identified in 1 89 samples showing no evidence of cervical cytological abnormality. Conclusions-Real time PCR is a rapid, efficient method for the detection of HPV with the separation of HPV-16 and HPV-18 on the basis of differential T-m. Preliminary results suggest it could prove useful if HPV testing is ad ded to cervical screening programmes.